Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Two In Vitro studies and one in vivo study has been conducted on the test material. All three studies show the same results and in all three studies the test material was considered to be non genotoxic. The test item material was considered to be non-mutagenic under the conditions of an Ames test.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 28th February 2012 and 11th December 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
Sufficient albino Hsd: ICR (CD-1®) strain mice were obtained from Harlan Laboratories UK Ltd., Oxon, UK. At the start of the main test the mice weighed 21 to 30g and were approximately six to ten weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.

The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding. Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent Diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.
Route of administration:
intraperitoneal
Vehicle:
The vehicle was supplied as a 10x concentrated solution by Gibco, as follows:

Supplier's identification : Dulbecco's Phosphate Buffered Saline Solution
Supplier's lot number: 938281
In-house serial number : V-5241
Date received : 02 November 2011
Expiry date : 01 May 2013
Storage conditions : Room temperature





Details on exposure:
A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The range-finding toxicity test was also used to determine if the main test was to be performed using both sexes or males only. With no evidence of toxicity via the oral route, and following discussions with the Sponsor, it was considered necessary to investigate the intraperitoneal route of administration to maximise exposure of the test item.

Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
Groups, each of seven male mice, were dosed once only via the intraperitoneal route with the test item at 2000, 1000 or 500 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 2000 mg/kg was killed after 48 hours.
Post exposure period:
Immediately following termination (i.e. 24 or 48 hours following dosing), the animals were dissected and the slide preparation had begun.
Remarks:
Doses / Concentrations:
2000, 1000 or 500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
7 males per dose group (2000, 1000 or 500 mg/kg)
Two additional groups of male mice were included in the study; one group (seven mice) was dosed via the intraperitoneal route with the vehicle alone (PBS).
A second group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control item was supplied by Acros Organics, as follows:

Supplier's identification: Cyclophosphamide
Supplier's lot number : A0302605
In-house serial number : R-5359
Date received : 27 April 2012
Expiry date : 27 April 2014
Storage conditions: Approximately 4°C in the dark

For the purpose of this study the positive control item was freshly prepared as required as a solution at the appropriate concentration in distilled water.




Tissues and cell types examined:
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each surviving animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension.
Details of tissue and slide preparation:
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each surviving animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. Where possible, the incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.

A positive mutagenic response would be demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group.

If these criteria were not fulfilled, then the test item would be considered non-genotoxic under the conditions of the test.

A positive response for bone marrow toxicity would be demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.

Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √(x +1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 to 2000 mg/kg
- Clinical signs of toxicity in test animals: In animals dosed with the test item at 2000 mg/kg via the oral route, clinical signs were not observed in any of the animals. Therefore, with no evidence of toxicity the intraperitoneal route of administration was investigated to maximise exposure of the test item. Using the intraperitoneal route, the clinical signs hunched posture, ptosis, increased activity, and irritation and over grooming of the injection site were observed in animals dosed with the test item at and above 1000 mg/kg.
- Evidence of cytotoxicity in tissue analyzed: No
- High dose with and without activation: There was no mutagenic response in the highest dose group either with or without metabolic activation.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):The test item was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.
- Ratio of PCE/NCE (for Micronucleus assay): Statistically significant decreases in the PCE/NCE ratio were not observed in any of the test item dose groups when compared to the vehicle control group. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.
- Statistical evaluation: There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Mortality Data and Clinical Observations:

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs were observed in animals dosed with the test item at and above 1000 mg/kg in both the 24 and 48-hour dose groups, where applicable, and included hunched posture and ptosis.

Summary of group mean data

Treatment group

Number of PCE with micronuclei per 2000 PCE

PCE/NCE Ratio

Group mean

SD

Group mean

SD

Vehicle control (PBS) 10 ml/kg

24-h sampling time

1.0

1.2

0.84

0.27

Positive control (Cyclophosphamide) 50 mg/kg

24-h sampling time

23.0***

8.4

0.95

0.20

Sodium glucoheptonate 2000 mg/kg

48-h sampling time

2.0

1.6

1.27

0.47

Sodium glucoheptonate 2000 mg/kg

24-h sampling time

2.1

2.9

1.56

0.43

Sodium glucoheptonate 1000 mg/kg

24-h sampling time

0.9

1.6

1.91

0.94

Sodium glucoheptonate 500 mg/kg

24-h sampling time

0.6

0.5

1.47

0.31

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

*** = P < 0.001

Conclusions:
Interpretation of results (migrated information): negative
The test item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

Introduction:

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Mammalian Erythrocyte Micronucleus Test", Method B12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

Methods:

A range-finding test was performed to find suitable dose levels of the test item, route of administration, and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. Following discussions with the Sponsor, the micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum recommended dose (MRD) of 2000 mg/kg and with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Additional groups of mice were given a single intraperitoneal dose of phosphate buffered saline (PBS) (7 male mice) or dosed orally with cyclophosphamide (5 male mice), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed after 24 hours.

Results:

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs were observed in animals dosed with the test item at and above 1000 mg/kg in both the 24 and 48-hour dose groups, where applicable, and included hunched posture and included hunched posture and ptosis.

Statistically significant decreases in the PCE/NCE ratio were not observed in any of the test item dose groups when compared to the vehicle control group. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test item was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Conclusion:

The test item was considered to be non-mutagenic under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro Studies

Ames

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, Sodium Glucoheptonate, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver 89 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Chromosome Aberration

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final Concentration of test item (µg/ml)

4(20)-hour without S9

88.75, 177.5, 355, 710, 1420, 2840

4(20)-hour with S9 (2%)

88.75, 177.5, 355, 710, 1420, 2840

24-hour without S9

88.75, 177.5, 355, 710, 1420, 2840

4(20)-hour with S9 (1%)

88.75, 177.5, 355, 710, 1420, 2840

The test item was only moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range which exceeded the maximum recommended dose level. The maximum dose level tested was based on the molecular weight of the hydrated form of the test material. It was subsequently discovered that when the test material is in solution, as in the sample tested, the water of crystallisation is lost and the molecular weight is therefore reduced. The test item was consequently over tested at 11.44 mM which was beyond the maximum recommended dose level (10 mM) using the molecular weight of the dihydrate form of the test material. However, since there was no response from the test item at any dose level in any of the exposure groups this was considered to be acceptable and have no impact on the study integrity.

In vivo Study

A range-finding test was performed to find suitable dose levels of the test item, route of administration, and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum recommended dose (MRD) of 2000 mg/kg and with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs were observed in animals dosed with the test item at and above 1000 mg/kg in both the 24 and 48-hour dose groups, where applicable, and included hunched posture and included hunched posture and ptosis.

Statistically significant decreases in the PCE/NCE ratio were not observed in any of the test item dose groups when compared to the vehicle control group. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

Justification for classification or non-classification

The test item material was considered to be non-mutagenic under the conditions of an Ames test, non-clastogenic to human lymphocytes in vitro and was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice, and therefore considered to be non-mutagenic in vivo.

Based on the above results the test material is considered to be non classified for genotoxicity according to DSD and CLP.